A phase I clinical trial of replication-competent Ad4-HIVenv and Ad4-HIVmosaic gag vaccines was initiated in the NIH clinical center in collaboration with NIAID and Dr. Mark Connors as Principal Investigator. The study is evaluating the safety and immunogenicity of the vaccines formulated as enteric coated capsules for oral administration and as a liquid for administration to the upper respiratory tract. At the same time, new replication-competent Ad vectors are under development for future clinical use following pre-clinical evaluation in the rhesus macaque model. Studies in mice and a pilot study in rhesus macaques showed that Ad-HIV envelope recombinants with deletions of E1B55K and/or E4orf1-4 early region genes not only had greater carrying capacity but also exhibited enhanced cellular and humoral immunity. A further pilot study in macaques will repeat these initial results and also examine a recombinant with an E1B55K deletion along with an E4orf 1 deletion. This pilot study will be prelude to a larger study in which the optimal vector will be used in a pre-clinical vaccine study evaluating overall immunogenicity and protective efficacy. A current pre-clinical study in macaques is evaluating a novel vaccine/microbicide regimen. Microbicides have been shown to be effective in preventing HIV transmission, however, their effectiveness depends on appropriate use which is often compromised by human behavior. Combining a prophylactic vaccine with microbicide use might provide protection against infection in instances when microbicides are not used properly. Therefore, we are immunizing rhesus macaques with our Ad-recombinant priming/Env protein boosting regimen in order to elicit mucosal immunity. Subsequently, we will expose the vaccinated animals to infectious SIV following administration of a microbicide. The microbicide (SAMT-247) is a zinc finger inhibitor that results in expression of non-infectious viral particles which nevertheless have intact envelope on their surface. We have postulated that the non-infectious particles will boost the immune response already elicited by the vaccine regimen. Overall, we are testing the hypothesis that the combined vaccine plus microbicide regimen is beneficial by providing either additive or synergistic protection against SIV infection. We have recently reported the development of a new technology for sorting and characterization of HIV/SIV from clinical specimens using nanoFACS. The virions can be stained and sorted based on either cellular antigens incorporated into the virion particles or alternatively using specific antibodies to different envelope epitopes. The ability to sort infectious HIV from clinically relevant samples provides material for detailed molecular, genetic, and proteomic analyses applicable to future design of vaccine antigens and potential development of personalized treatment regimens.